There is a demand for a sensitive and selective method for in vitro and in vivo medical diagnostics and treatment of disease at the molecular level. In addition, there is a demand for a rapid, simple, cost-effective technique for screening air, water and biological samples, such as blood, saliva, bodily fluid, cells, tissues and organs to identify various components therein. Screening can involve detection of biochemical and biological species such as harmful chemicals, bacteria, viruses, defected genes, proteins, metabolites and biomarkers of diseases in organs, tissues and cells. Furthermore, there is a need for early detection of biological and chemical warfare agents for homeland defense.
Screening can also be used to identify the presence or absence of medical diseases and infectious pathogens. Regarding biological fluids, tissues, and organs, the use of inexpensive screening analyses can allow the rapid detection and improved treatments of many illnesses. Rapid and effective medical screening tests can also reduce the cost of health care by preventing unnecessary and generally more costly reactive medical treatments.
A critical factor in many diagnostics is the rapid, selective, and sensitive detection of biochemical substances, such as proteins, metabolites, nucleic acids, biological species or living systems, such as bacteria, virus or related components at ultra-trace levels in samples, tissues or organs of interest. In the case of medical diagnostic applications, biological samples can include tissues, blood and other bodily fluids. To achieve the required level of sensitivity and specificity in detection, it is often necessary to use a method that is capable of identifying and differentiating between a large number of biochemical constituents in complex mixed samples.
Living systems possess exquisite recognition elements often called bioreceptors, such as antibodies, proteins, enzymes and genes, which allow specific identification and detection of complex chemical and biological species. Molecular probes, which comprise a bioreceptor for molecular recognition and binding and a label for detection, exploit this powerful molecular recognition capability of bioreceptors. Due to the high level of specificity of the DNA hybridization process, there is an increasing interest in the development of DNA bioreceptor-based molecular probe systems. Applications for these systems include infectious disease identification, medical diagnostics and therapy, biotechnology and environmental bioremediation.
Nucleic acid based molecular probe systems can be designed using simple rules to recognize and detect a wide variety of targets with almost any desired degree of specificity. Moreover, such probe-based systems can be chemically produced with relative ease. Synthetic oligonucleotides can be made specific for desired sequences by varying length, sequence, and hybridization conditions of the probe oligonucleotide to permit identification and quantification of the presence of its complementary sequence within a heterogeneous mixture.
Peptide Nucleic Acid (PNA) is a system similar to DNA in which the backbone is a pseudopeptide rather than a sugar. PNA functions in a manner similar to DNA and binds to complementary nucleic acid strands. The neutral backbone of PNA often leads to stronger binding and greater specificity than normally achieved with DNA. In addition, the unique chemical, physical and biological properties of PNA have been exploited to produce powerful molecular probes. Important new applications have emerged that could not be performed using oligonucleotides.
There has been recent research and development relating to molecular probes and related detection systems called biosensors. One type of biosensor device, often referred to as a “biochip,” applies spectroscopy using a semiconductor-based detection system and biotechnology-based probes. These probes have generally included luminescence labels, such as fluorescent or chemiluminescent labels for gene detection. Although sensitivities achieved by luminescence techniques are generally adequate for certain applications, alternative techniques with improved spectral selectivities are desirable to overcome the limited spectral specificity generally provided by luminescent labels.
Spectroscopy involves an analytical detection technique concerned with the measurement of the interaction of radiant energy with matter and with the interpretation of the interaction both at the fundamental level and for practical analysis. Interpretation of the spectra produced by various spectroscopic instrumentation has been used to provide fundamental information on the atomic and molecular energy levels, the distribution of species within those levels, the nature of processes involving change from one level to another, molecular geometries, chemical bonding, and interaction of molecules in solution. Comparisons of spectra have provided a basis for the determination of qualitative chemical composition and chemical structure, and for quantitative chemical analysis.
Vibrational spectroscopy is a useful technique for characterizing molecules and for determining their chemical structure. The vibrational spectrum of a molecule, based on the molecular structure of that molecule, is a series of sharp lines which constitutes a unique fingerprint of that specific molecular structure. If the vibrational spectrum is to be measured by an optical absorption process, optical fibers deliver light from a light source to a sample, and after passage of the light through the sample, the optical signal generated by the exciting optical energy is collected. This collected light is directed to a monochromator equipped with a photodetector for analyzing its wavelength and/or intensity.
One particular spectroscopic technique, known as Raman spectroscopy, utilizes the Raman effect, which is a phenomenon observed in the scattering of light as it is reflected by a material medium, whereby the light experiences a change in frequency and a random alteration in phase. When light is scattered from a molecule, most photons are elastically scattered. The scattered photons have the same energy (frequency) and, therefore, wavelength, as the incident photons. However, a small fraction of light (approximately 1 in 107 photons) is scattered at optical frequencies different from, and usually lower than, the frequency of the incident photons. The process leading to this inelastic scatter is termed the Raman effect. Raman scattering can occur with a change in vibrational, rotational or electronic energy of a molecule.
The difference in energy between the incident photon and the Raman scattered photon is equal to the energy of a vibration of the scattering molecule. A plot of intensity of scattered light versus energy difference is a Raman spectrum. The wavelengths present in the scattered optical energy are characteristic of the structure of the molecule, and the intensity of this optical energy is dependent on the concentration of these molecules.
Numerically, the energy difference between the initial and final vibrational levels, ν, or Raman shift in wavenumbers (cm−1), is calculated through equation 1 below:ν=(1/λincident)−(1/λscattered)  (1)
Where λincident and λscattered are wavelengths (in cm) of the incident and Raman scattered photons, respectively. The vibrational energy is ultimately dissipated as heat. Because of the low intensity of Raman scattering, heat dissipation does not cause a measurable temperature rise in the material.
Raman spectroscopy is complementary to fluorescence, and has been used as an analytical tool for certain applications due to its excellent specificity for chemical group identification. However, low sensitivity historically has limited its applications. Recently, the Raman technique has been rejuvenated following the discovery of a Raman enhancement of up to 106 to 1010 for molecules adsorbed on microstructures of metal surfaces. The technique associated with this phenomenon is known as surface-enhanced Raman scattering (SERS) spectroscopy. The enhancement is due to a microstructured metal surface scattering process which increases the intrinsically weak normal Raman scattering (NRS) due to a combination of several electromagnetic and chemical effects between the molecule adsorbed on the metal surface and the metal surface itself.
The enhancement is primarily due to plasmon excitation at the metal surface. Thus, the effect is generally limited to Cu, Ag and Au, and to a few other metals for which surface plasmons are excited by visible radiation. Although chemisorption is not essential, when it does occur there may be further enhancement of the Raman signal, since the formation of new chemical bonds and the consequent perturbation of adsorbate electronic energy levels can lead to a surface-induced resonance effect. The combination of surface- and resonance-enhancement, often referred to as surface-enhanced resonance Raman scattering (SERRS) can occur when adsorbates have intense electronic absorption bands in the same spectral region as the metal surface plasmon resonance, yielding an overall enhancement as large as 1010 to 1012.
Nanoparticles can comprise solid metal of nanoscale size or nanoparticles coated with metal layers. Nanospheres of dielectric materials coated with a thin layer of silver (metal nanoshell) have been found to be SERS active. Nanospheres of magnetic materials coated with a thin layer of metal can also be used as SERS-active magnetic nanoparticles. The core diameter and the metal thickness of nanoshells can be varied to modify the SERS properties of the nanoparticles as disclosed in an article co-authored by the inventor. [R. L. Moody, T. Vo-Dinh, and W. H. Fletcher, “Investigation of Experimental Parameters for Surface-Enhanced Raman Spectroscopy,” Appl. Spectrosc., 41, 966 (1987)].
A useful non-Raman based assaying techniques using stem-loop oligonucleotide probes has been disclosed. These probes are referred to as “molecular beacons” and were first disclosed as providing a rapid, quantitative assay technique by Tyagi and Kramer (Tyagi, S., F. R. Nature Biotechnology, 14, 303-308 (1996)). Molecular beacons are designed to have loop sequences which are complementary to a target nucleic acid (e.g., rRNA). The loop sequence is disposed between a first and a second stem sequence, the respective stem sequences being complements of one another. The molecular beacon includes a fluorescent molecule on the end of the first stem and a quenching molecule on the end of the second stem.
In the absence of the complementary target sequence the fluorescence upon irradiation remains low (quenched) due to physical proximity between the fluorophore and the quencher. When the complementary sequence is present, the loop opens and the fluorophore and the quencher separate so that they are no longer in physical proximity, so that the molecular probe generates relatively strong fluorescent signal upon irradiation when target nucleic acids are present.